There is a need in the art for methods of making and using modified oocytes. For example, there is a need in the art for methods of making and using modified oocytes as a means for avoiding or eliminating the inheritance of deleterious mutations of the mitochondrial genome. Mitochondrial DNA mutations transmitted maternally within the oocyte cytoplasm often cause life-threatening disorders. A critical determinant of the phenotypic severity in most maternally inherited mitochondrial diseases is heteroplasmy, i.e. the proportion of mutant, relative to total, mitochondrial DNA (mtDNA) within a cell. Due to the cytoplasmic segregation of mitochondria during cell division, the level of heteroplasmy is subject to broad fluctuations, in particular during the developmental expansion of mtDNA from the premeiotic germ cell to the mature human oocyte. As a result, an unaffected carrier of a mtDNA mutation may have an affected child. Whilst prenatal genetic diagnosis can select embryos with a reduced mutation load, variation between blastomeres in single embryos limits the effectiveness of such screening, and significant levels of mutant mtDNA can remain resulting in a carrier. Because of such issues, the Nuffield Council on Bioethics has endorsed research to prevent transmission of mtDNA mutations, including the transfer of the nuclear genome of one oocyte into an enucleated oocyte containing normal mitochondria.
Another area in which there is a need in the art for methods of making and using modified oocytes is in the area of fertility treatment. In vitro fertilization (“IVF”) treatments fail at increased rates as maternal age increases, at least in part because the developmental potential of oocytes decreases with advancing maternal age. In women above the age of forty-two with the ability to hyperstimulate, the IVF success rate falls to less than 5% of live births and miscarriage rates approach 50%, with a high aneuploidy rate. This is both a significant clinical problem as well as source of personal suffering to prospective parents. The causes of this drop in fertility include a decreased quality of the oocytes. Oocytes are less likely to be fertilized, are more likely to contain aneuploidies, less likely to implant (Piette et al. 1990), and more likely to result in spontaneous abortion or congenital abnormalities. They are also less in number. Many of the developmental defects arise as a consequence of abnormal chromosome segregation in mitoses of preimplantation stage embryos, and at the first and second meiotic division. The number of oocytes obtained from women of advanced maternal age is often also reduced (Piette et al. 1990), thereby further reducing the chance of a successful pregnancy.
A decline in developmental potential also occurs when oocytes remain unfertilized for prolonged periods either in vitro or in vivo: the timing for optimal fertilization and development in mice is less than 12 hours post ovulation (see, for example, Woods et al. 1990; Wakayama et al. 2004; Ono et al. 2011), and in humans is within 4 to 12 hours after ovulation or oocyte retrieval (see, for example, Morton et al. 1997; Chen and Kattera 2003). In vitro postovulatory aging of oocytes occurs during prolonged in vitro culture of oocytes before fertilization, and is a clinically important issue. Failure to fertilize oocytes occurs in approximately 5% to 10% of conventional IVF cycles, and on average 15-30% of oocytes remain unfertilized even with fully functional sperm (see, for example, Barlow et al. 1990; Nagy et al. 1993). Generally oocytes that have not been successfully fertilized in an IVF cycle cannot be used in subsequent IVF cycles. The rate of pregnancy and implantation rates decreases to 5-20% and 1.7%, respectively, at 62 hours post human chorionic gonadotropin (hCG) treatment when oocytes are aged in vitro, while the corresponding rates are 48.0% and 20.2%, respectively, at 46 hours post-hCG treatment (Yuzpe et al. 2000; Chen and Kattera 2003).
Approximately 10-30% of oocytes are immature at retrieval from the ovary, at either the metaphase I or the germinal vesicle stage. While about half of them mature to the MII stage within hours after retrieval, and are useful for IVF treatments, many oocytes remain arrested at the germinal vesicle stage. And those that can be matured to the metaphase II stage often have a reduced developmental competence compared to oocytes that are already mature when they are retrieved from the ovary. This is why immature oocytes are generally not used for IVF treatments and instead are discarded. The loss of these oocytes reduces the chance of successful IVF treatments, especially for women of advanced age, who may start out with a lower total number of retrieved oocytes. Failure to mature to the MII stage can also be a medical condition, where all oocytes fail to mature, resulting in infertility (Chen et al. 2010).
Currently, after exhausting other available options, women who are unsuccessful with fertility treatments, such as in vitro fertilization treatments, using their own eggs sometimes resort to using oocyte donors to achieve a pregnancy—even though the children born from such pregnancies are not genetically related to the mother. There is a need in the art to develop a better understanding of the molecular events that lead to reduced developmental potential of oocytes with maternal age, and also with increased culture time in vitro, and a need for new methods that could improve the developmental potential of such oocytes, allow rescue of oocytes from failed IVF treatments, and give women who have been unable to achieve pregnancy using traditional in vitro fertilization methods another chance at becoming pregnant with a child that is genetically related to her.